1. Field of the Invention
The present invention relates to oligonucleotide sequences specific to Helicobacter pylori urease and useful as DNA probes and primers in the detection of H. pylori infections in humans.
2. Discussion of the Background
Helicobacter pylori (also described by the expression H. pylori) is a Gram negative bacterium found exclusively nowadays at the surface of the stomach mucosa in man, and more particularly around the lesions of the craters due to gastric and duodenal ulcers. This bacterium was initially called Campylobacter pyloridis (Warren et al. (1983) Lancet 1. 1273-1275).
Like most bacteria, H. pylori is sensitive to a medium of acidic pH but can however tolerate acidity in the presence of physiological levels of urea (Marshall et al. (1990) Gastroenterol. 99:697-702). By hydrolysing urea to carbon dioxide and ammonia which are released into the microenvironment of the bacterium, the urease of H. pylori is assumed to permit the survival of the bacterium in the acidic environment of the stomach. Recently, studies conducted on animal models have provided elements suggesting that urease is an important factor in the colonization of the gastric mucosa (Eaton et al. (1991) Infect. Immun 59:2470-2475). Urease is also suspected of causing injury either directly or indirectly to the gastric mucosa.
Helicobacter pylori (H. pylori) is presently recognized as the etiological agent of antral gastritis, and appears to be once of the cofactors required for the development of ulcers. Furthermore it seems that the development of gastric carcinomas may be linked to the presence of H. pylori.
All of the strains isolated in the clinic from biopsies or gastric juice synthesize a very active urease, which is exposed at the surface of the bacterium and is one of the most immunogenic proteins of H. pylori. The urease is suspected of playing a role in the pathogenic process, a fact which has been confirmed by experiments performed on the pig which show that weakly producing urease strains obtained by chemical mutagenesis were incapable of colonizing the stomach of the pig. These results obtained after chemical mutagenesis do not make is possible to attribute with certainty the diminution of urease production to an inability to colonize the stomach, since other genes may be inactivated during generalized mutagenesis. Hence these are not controllable mutations and, consequently, this procedure has no real value in the creation of agents designed to diminish, and even prevent, the harmful effects of urease in the case of an infection by H. pylori.
In addition to this role in the colonization of the stomach, it has been shown that urease as well as the ammonia released might have a direct cytotoxic effect on epithelial cells and an indirect effect by inducing an inflammatory response which might be responsible for the gastric lesions.
The urease is hence one of the most important determinants of pathogenicity and the construction of isogenic strains of H. pylori specifically inactivated in the genes responsible for the expression of urease, whether they be structural genes or accessory genes, are of primary importance for defining the role of urease in the colonization step, and for use in the construction of strains which can be used to protect individuals in a vaccination process, for example by the construction of attenuated strains.
Hitherto the urease genes had been localized on a 34 kb fragment of the H. pylori chromosome and had been associated with a 4.2 kb region present in this fragment. Four genes designated by the terms ureA, ureB, ureC and ureD had been associated with this region of 4.2 kb. This region led to the production of a urease-positive phenotype when the DNA of 4.2 kb was transferred by the intermediary of a shuttle vector of Campylobacter jejuni.
However, the transformation of E. coli cells with the DNA of 4.2 kb previously described did not lead to the expression of urease activity in E. coli.